This invention pertains generally to biosensors and more particularly to biosensors made by incorporating membrane proteins into polymerized lipid bilayers.
Biological cells have proteins in the cell membrane which react with molecules or other stimuli from outside the cell. The proteins sense the presense of an external stimulus and change the characteristics of the cell membrane, typically making the membrane more or less permeable to external molecules or ions. These proteins, therefore, function as biosensors for the cell by sensing the presence of stimuli in the cell's environment.
Many biophysical techniques have been developed to study the proteins which make up the membranes of biological cells. Among the most powerful of these techniques has been the isolation of membrane proteins, followed by reincorporation of the purified polypeptide into a synthetic membrane system in which the function can be studied in the absence of the frenetic activity which characterizes most native membrane preparations.
Reincorporation of proteins into synthetic membranes has most often been into vesicular membrane systems since they form spontaneously, provide an inside and outside aqueous space to probe, and can be stable for a long time. However, when access to the electrical activity of a membrane protein in a reconstituted system is required, most reconstituting vesicular membrane systems have compartments which are too small for convenient insertion of recording microelectrodes. The "black lipid membrane", or BLM, has offered an alternative solution. It allows transfer of protein from vesicles into a planar lipid bilayer separating two large aqueous compartments into which electrodes may be inserted for electrical measurement.
Montal and Mueller developed a method to form bilayers from monolayers on the surface of water. M. Montal and P. Mueller, Proc. Nat. Acad. Sci. U.S.A., 69, 3561-3566 (1972). This method for making bilayers from monolayers (BFM) allows formation of bilayers in the absence of organic solvents which contaminate an otherwise cleanly-reconstituted system. As it is usually practiced, the method enables one to produce a bilayer patch half a millimeter or so in diameter. This technique is, however, frustrating even for its most avid advocates, and additionally, once formed, the bilayer is only stable for a few minutes or hours at the most.
Recently, a new method, the patch clamp technique, was developed. O. P. Hamill, A. Marty, E. Neher, B. Sakmann, F. J. Sigworth, Pflugers Arch., 391, 85-100 (1981). In the patch clamp technique, a glass micropipette with a tip diameter of about 2 micrometers is placed against an existing bilayer or is moved through an air-water interface on which a monolayer exists. R. Coronado and R. Latorre, Biophys. J., 43, 231-236 (1983). This technique results in the formation and sealing of a bilayer across the pipette tip. The resulting bilayer can have a resistance in excess of 10 gigaohms. Such a seal allows the recording of the current flow through single ion channels with time response in the sub-millisecond range. This technique has the major advantage over the BLM and BFM techniques that it is much easier to form bilayers on the glass pipette tips. Although the lifetime of bilayers on the pipettes may be no longer than that in the BLM and BFM techniques, the ability to pull off the old pipette and put on a new one and immediately get a new working patch without having to clean out the apparatus makes the patch pipette technique far easier to use as a research technique. It has recently been shown that the glass of the pipette can be used to form good seals to artificial lipid monolayers on water, from which good patches form with useful electrical properties.
Many different membrane proteins have been monitored electrically using the above techniques. Among these are various neurotransmitter receptors, such as the acetylcholine receptor, visual proteins such as rhodopsin, and the bacterial proton pump bacteriorhodopsin. It has also been shown that sensory tissue, such as olfactory tissue, when presented to a BLM in such a manner that some of it is reincorporated into the bilayer, can induce an odor-dependent current flow across the membrane. V. Vodyanoy and R. B. Murphy, Science, 220, 717-19 (1983).
The generally short life of bilayers formed by all of the above-mentioned techniques has been an experimental annoyance that has hampered progress in the development of biosensors. Sensors produced with the above techniques would be easy to disrupt, mechanically limited to use in the laboratory, and have a short life span.
A lipid membrane is, therefore, needed which can be produced quickly and easily and overcome the poor longevity and durability of BLMs, BFMs, and patches. Improvement in the longevity and durability of the lipid membrane would provide a practical sensor which could be used in the laboratory and in the field.